Denaturant-Free Electrophoresis of Biological Molecules Under High Temperature Conditions

ABSTRACT

The present invention relates to a method of separating a sample comprising biological compounds, such as nucleic acids. The nucleic acids are subjected to electrophoresis using a matrix that is essentially free of denaturants and having at least one random, linear copolymer comprising a first comonomer of acrylamide and at least one secondary comonomer. A temperature of at least a portion of the matrix is at least about 80° C.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/661,558,filed Sep. 15, 2003, which is a continuation of application Ser. No.10/258,547, filed Oct. 25, 2002, now abandoned, which is a nationalphase application of application no. PCT/US01/13336, filed Apr. 25,2001, which claims priority to U.S. Provisional Application No.60/199,389, filed Apr. 25, 2000, which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to methods and devices for sequencingnucleic acids in separation matrices that are essentially free ofchemical denaturants.

BACKGROUND

A conventional DNA sequencing gel matrix typically contains 3-9 M urea,or a combination of urea and formamide as a denaturant. The function ofa denaturant in a gel is to help keep DNA molecules denatured duringelectrophoresis in order to achieve accurate base calling. The existenceof urea or formamide in a gel matrix represents a distinctive differencebetween denaturing gel electrophoresis for DNA sequencing (separatingsingle-stranded DNA) and non-denaturing gel electrophoresis forseparating double stranded DNA. The denaturing power of a gel matrix isgenerally proportional to the concentration of a denaturant in the gelmatrix. Higher denaturing powers minimize compression, a self-foldingbehavior, of single stranded DNA fragments in a DNA sequencing sample.

When urea is used, however, the denaturing power is limited by thesaturation concentration of urea, which is about 9M. When formamide isused, there is also a limit, which is the manageable viscosity of amatrix and separation speed. For example, because the viscosity of thematrix increases significantly with the percentage of formamide in agel, separation speed decreases with higher percentages of formamide.Sometimes, the denaturing power of a gel with a maximum concentration ofurea or formamide still does not provide sufficient denaturing power toresolve some compression bands in GC-rich DNA samples.

A popular method to overcome the above-mentioned problem of insufficientdenaturing power is to heat up a gel during electrophoresis, typically35-70° C., and add a denaturant. The combination of high temperatureelectrophoresis and high concentration denaturant typically providessufficient denaturing power to resolve difficult compression bands.

There are several issues, however, associated with electrophoresis usinga matrix containing urea or formamide. First, urea and formamide degradein the basic solution that is typically used for DNA sequencing (pH8.0-8.5). Higher temperatures accelerate such degradation. Thedegradation of urea or formamide has adverse effects on the gel andseparation columns. The degradation products include ammonia, uric acid,and formic acid. These products increase the ion concentration and pH ofthe matrix. They may also cause bubble formation in a matrix at highertemperatures. When an uncoated capillary is used, these degradationproducts reduce the adhering affinity between the polymer dynamiccoating and the capillary wall. This allows electroosmotic flow tooccur, which consequently reduces separation efficiency. Capillarylifetime is also shortened because the decreased coverage of polymercoating on the capillary wall allows biomolecules to attack and adsorbonto the capillary wall, which in turn degrades separation efficiency.

To minimize these problems, one can take several approaches: a) optimizethe electric field strength and column temperature so that thedegradation products can be consistently driven out of the separationcolumn at a rate that is equivalent to or higher than the rate ofgeneration; b) develop better dynamic coating polymers that adsorb ontocapillary wall more efficiently under high temperature; c) reduce the pHvalue of the gel matrix, e.g., from pH 8.3 down to pH 7.6, in order toreduce the degradation rate of urea or formamide at high temperatures,and to enhance the adsorbing efficiency of polymer on the capillarywall. These methods, however, have limitations.

SUMMARY OF THE INVENTION

The present invention relates to a method of separating a mixture ofnucleic acids, comprising subjecting the biological molecules toelectrophoresis using a matrix that is essentially free of denaturants,the matrix having at least one random, linear copolymer comprising afirst comonomer of acrylamide and at least one secondary comonomer,wherein a temperature of at least a portion of the polymer matrix is atleast about 75° C. and more preferably at least about 80° C. The maximumtemperature of the matrix should be less than the boiling point of afluid within the matrix, but preferably is less than about 95° C. In oneembodiment, the matrix is completely free of denaturants.

The comonomers are preferably randomly distributed along the copolymer.At least one secondary comonomer is selected from the group consistingof vinyl monomers, monomers of acrylamide derivatives, monomers ofacryloyl derivatives, monomers of acrylic acid derivatives, monomers ofpolyoxides, monomers of polysilanes, monomers of polyethers, monomers ofderivatized polyethylene glycols, monomers of cellulose compounds, ormixtures thereof each having between 2-24 carbon atoms.

In one embodiment, the copolymer is polymerized using about a 1:1 ratioof acrylamide and another comonomer. The other comonomer is preferablyN,N-dimethylacrylamide monomer. The ratio of reactivity of the at leastone secondary comonomer to said primary comonomer is preferably between0.3 and 2.

In another embodiment, the matrix has a viscosity between 100 and 50,000Cp. The at least one linear random copolymer has a molecular weightbetween 100,000 and 5,000,000 Daltons. In a preferred embodiment thecompolymer comprises a buffer having a basic pH. Preferably, the buffercomprises about 89 mM Tris, 89 mM borate, and 2 mM EDTA. In a preferredembodiment, the buffer has a pH of at least about 8 and preferably fromabout pH 8 to pH 8.3.

In yet another embodiment, a second mixture of nucleic acids issubjected to electrophoresis within the matrix with at least a portionof the matrix being heated to at least about 75° C. preferably at leastabout 80° C. The second mixture of nucleic acids may be identical to theearlier electrophoresed mixture or the second mixture may be different.Preferably, the electrophoresis step can be repeated up to at leastabout 25 times so that about 25 mixtures can be electrophoresed withoutfirst providing a new matrix.

In another embodiment, a cooled portion of the matrix is cooled to lessthan about 25° C. The cooled portion is preferably disposed between adetection zone of the matrix and the heated portion of the matrix sothat the cooled portion receives nucleic acids from the heated portionof the matrix. The length and temperature of the cooled portion arepreferably selected to allow DNA that was denatured in the heatedportion to substantially re-anneal prior to being detected.

Another embodiment of the invention relates to a method ofelectrophoresing a plurality of mixtures of biological compounds,comprising subjecting a first mixture to electrophoresis using a matrixthat is essentially free of denaturing agents, the first mixturecomprising nucleic acids, and wherein a temperature of at least aportion of the matrix is sufficient to substantially denature thenucleic acids. During electrophoresis of the first mixture, thetemperature of the matrix preferably is between 80° C.-99° C. Morepreferably, the temperature is between 80° C.-95° C. and most preferablyis between 80° C.-90° C. The temperature of the matrix is insufficientto boil a fluid, such as water, present in the matrix and so thisdetermines the upper temperature limit, subject to the atmosphericconditions.

A second mixture is subjected to electrophoresis using substantially thesame matrix, the second mixture comprising a complex of at least twobiological compounds. By substantially the same matrix it is meant, forexample, that the same support is used to electrophorese both the firstand second mixtures without first replacing more than about 20% of thematrix present in the support. Preferably, less than about 5%, and morepreferably none of the matrix is replaced.

In a preferred embodiment, the complex comprises at least one of anucleic acid-protein complex and a protein-protein complex. It should beunderstood that the first and second mixtures can be electrophoresed ineither order.

Another embodiment of the present invention relates to a system forelectrophoretically sequencing at least one nucleic acid sample. Thesystem comprises at least one support suitable for retaining a matrix inwhich electrophoretic separation of nucleic acid samples may beconducted. A heat source is in thermal contact with the at least onesupport, the heat source being configured to heat at least a portion ofthe at least one support to at least about 80° C. The support preferablyprovides sufficient thermal contact between the heat source and thematrix retained by the support so that heating the support to at leastabout 80° C. also heats a portion of the matrix to at least about 80° C.

In one embodiment, the system further comprises a cooling deviceconfigured and arranged to cool a cooled portion of the at least onesupport, the cooled portion receiving samples from the heated portion ofthe support and being disposed between the heated portion and adetection zone of the capillary. The cooling device is preferablyconfigured to cool the temperature of the cooled portion of thecapillary to less than about 25° C.

In another embodiment, the support contains a matrix suitable forelectrophoretic separation of a nucleic acid sample, the matrix beingessentially free of denaturing agents.

Another embodiment of the present invention relates to a system forelectrophoretically sequencing a plurality of nucleic acid samples, thesystem comprising a plurality of capillaries, each capillary having afirst end, the first ends being arranged in a two-dimensional arraycorresponding to an array of wells of a microtitre tray, each of saidwells configured to contain at least one of the nucleic acid samples.The system includes an apparatus to fluidly associate each of saidnucleic acids samples with a respective first end to introduce thenucleic acid samples to the capillaries. Fluidly associating a samplewith the first end of the capillary with a sample in a well preferablyintroduces a sufficient quantity of the sample into the capillary toallow electrophoretic separation of the nucleic acids in the samplefollowed by detection of the separated nucleic acids.

The device includes a heat source in thermal contact with said pluralityof capillaries and configured to heat at least a heated portion of eachof said capillaries to a temperature of at least about 80° C., andcomputer means configured to operate the heat source to heat the heatedportions to at least about 80° C.

The system preferably includes a light source arranged to illuminatesaid samples and a detector arranged to detect fluoresced light emittedby said samples.

In one embodiment, the system further comprises a cooling deviceconfigured and arranged to cool a cooled portion of each of at leastsome of the capillaries, the cooled portions disposed to receive nucleicacids from the heated portions of the capillaries. The cooling device ispreferably configured to cool a temperature of each cooled portion toless than about 25° C.

In another embodiment, the capillaries contain a matrix suitable forelectrophoretic separation of a nucleic acid sample, the matrix beingessentially free of denaturing agents.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is described in detail below with reference to thedrawings in which:

FIG. 1 a & 1 b show two embodiments for a device utilizing thermaldenaturation in accordance with the present invention;

FIGS. 2 a and 2 b show the result of sequencing a PGEMI/U sample using a5% copolymer gel in 1xTBE, 7M urea, at room temperature;

FIG. 3 shows a graph of Phred score versus called bases for theseparation of FIGS. 2 a and 2 b;

FIGS. 4 a-4 c show the result of sequencing a PGEM/U sample using 5%copolymer gel in 1xTBE with no urea, at 80° C. according to the presentinvention; and

FIG. 5 shows the Phred score v. called bases for the separation of FIGS.4 a and 4 b.

DETAILED DESCRIPTION OF THE INVENTION Matrix for Electrophoresis

The present invention overcomes the problems associated with thepresence of denaturing agents such as, for example, urea or formamide,in a medium configured to support the electrophoretic separation ofbiological molecules. Preferably, the medium is configured to supportthe electrophoretic sequencing of nucleic acids such as DNA undertemperature conditions sufficient to at least partially denature thenucleic acids. The medium is preferably a matrix, such as a sievingmatrix, that is essentially free of urea, formamide, or other denaturingagents. As used herein, the term matrix is synonymous with the term gel,which is often used to describe media used for electrophoreticseparations.

The matrix of the present invention may be used with any suitableelectrophoresis format, such as, for example, slab gel electrophoresis,capillary electrophoresis, or microchip electrophoresis. Preferably, thematrix is retained by a support that, together with the matrix, definesa path through which a sample migrates. Suitable supports include, forexample, plates for retaining slab gels, a capillary, or microchipchannel. The support is preferably coated or otherwise modified tominimize electroosmotic flow, as understood in the art. Modifiedsupports include supports that are formed materials, such as plastics orother polymers, that themselves minimize electroosmotic flow.Alternatively, the matrix itself can provide a “coating” functionreducing the amount of electroosmotic flow. It should be understood,however, that the present invention is suitable for use with supportsthat are either unmodified or uncoated such that electroosmotic flowoccurs.

By essentially free of denaturing agents it is meant that denaturingagents, if any, are present in the matrix in an amount such that theagents themselves or degradation products of the agents do not adverselyaffect the matrix or separation support. For example, separationsupports are typically modified or configured to minimize electroosmoticflow during sequencing. The denaturing agents, if present, arepreferably present in an amount that is insufficient to increaseelectroosmotic flow by an amount sufficient to degrade separationperformance. More preferably, the essentially denaturant free matrix ofthe present invention is completely free of urea, formamide, and otherdenaturing agents.

Because the denaturing power of the present matrix preferably dependsupon the temperature of the matrix, rather than the presence of adenaturing agent, the same matrix can be operated in denaturing mode forone sample and in non-denaturing mode for a successive sample. Theability to alternate the same matrix between denaturing andnon-denaturing modes advantageously increases the rate at whichsuccessive samples can be analyzed because time consuming capillaryflushing steps are not required to remove or add the denaturing agent tothe matrix or to change the matrix itself. For example, the matrix ofthe present invention may be used to separate a first sample ofdouble-stranded DNA, without thermal denaturing, and to separate asecond sample of DNA in the form of single-stranded DNA using thermaldenaturing, as in DNA sequencing.

The matrix of the present invention also allows binding assays to beperformed in the same matrix that can be used for sequencing DNA withdenaturing. For example, electrophoretic assays of DNA-proteininteractions or assays of protein-protein interactions require that thetemperature of the matrix be raised sufficiently high to induceelectrophoretic mobility differences in the DNA-protein orprotein-protein complex. The present matrix, being essentially free ofdenaturing agents, allows the same matrix to be operated at temperaturessufficiently high to perform a binding assay on one mixture and sequenceDNA with thermal denaturing in a second mixture. Of course, by reducingthe temperature, the same matrix can be used to separate mixtureswithout denaturing. As used herein, the term mixture refers to a samplecomprising compounds to be separated, sequenced, or otherwise assayed todetermine a property of a compound present in the sample. Assays includeboth qualitative and quantitative determinations.

The matrix of the present invention can also be used in the quantitationor quality control of products formed during a polymerase chain reaction(PCR). The PCR products can be very large, such as greater than about1000 basepairs. The matrix of the present invention can be operated witha viscosity sufficiently low to facilitate separation of the large PCRproducts. In order to compensate for the loss of denaturing power due tobeing essentially free of denaturants, the temperature of at least aheated portion of the matrix is preferably sufficient to denaturesubstantially all of the DNA to be sequenced. The heated portionpreferably comprises substantially all of the migration distance, whichis the distance along the migration path between the region where themixture is introduced into the matrix and the region where components ofthe mixture are detected. For example, the heated portion of the matrixcomprises at least 50%, preferably at least 75%, more preferably atleast 85%, of the migration distance. Thus, during electrophoresis, DNAin a mixture preferably remains denatured for substantially all of themigration time. Preferably, the DNA remains denatured for at least 50%,of the time required to migrate from the injection region to thedetection region.

The temperature of the heated portion is at least about 75° C. andpreferably is between 80° C.-99° C. More preferably, the temperature isbetween 80° C.-95° C. and most preferably is between 80° C.-90° C.

The polymer of the matrix of the present invention may be any polymerthat is suitable for use in electrophoresis and is able to be operatedat temperatures sufficient to denature at least a portion of the DNA.

Preferably, the polymer is a copolymer formed of a 1:1 ratio ofacrylamide and N,N-dimethylacrylamide (DMA) monomer. The matrix of thepresent invention preferably contains from about 0.2 to about 10%copolymer by weight. More preferably, the amount of copolymer is fromabout 1 to 6%, with about 5% most preferred. Polymerization techniquessuitable to produce polymers of the present invention and other polymerssuitable for use in the present invention, are described ininternational application no. PCT/US00/00793 published Jul. 20, 2000 aspublication number WO 0042423, which is incorporated herein by referencein its entirety.

In a preferred embodiment, the matrix comprises at least one randomcopolymer forming a targeted linear copolymer. The random copolymerpreferably includes more than one monomer, with different monomer unitsbeing distributed along the copolymer chain in no specific pattern.Preferably, the copolymer is not crosslinked with other copolymers. Therandom copolymers are preferably composed of at least two or morecomonomer types. The ratio of comonomers can be continuously adjusted tooptimize the properties for electrophoretic separation. The ratio ofcomonomers may be any ratio that provides the desired properties of therandom copolymer. The comonomers must be sufficiently water soluble tobe used in an electrophoretic separation. Typically, there is a primarycomonomer that gives the random copolymer chain its primary physical,chemical, and sieving properties. Preferably, the primary comonomer isan acrylamide or an acrylamide derivative, which contains between 3-24carbon atoms, is either saturated or unsaturated, and is eithersubstituted or unsubstituted.

Examples of suitable acrylamide derivatives include, but are not limitedto, N,N-dimethacrylamide, N,N-dimethylmethacrylamide,N-ethylmethacrylamide, N-ethylacrylamide, N-methylacrylamide,N-methylmethacrylamide, and methacrylamide. The primary comonomers areavailable commercially or by simple derivatization of monomer units.

The secondary comonomers are selected for their inherent properties thatmay be incorporated into the copolymer chains. These inherent propertiesinclude, but are not limited to, one or more of hydrophilicity,hydrophobicity, self coating properties, copolymer chain backbonestiffness, stability of copolymer entanglement structure at differenttemperature and electric fields, resistance to hydrolysis, processivityof copolymer chain extension, gel matrix viscosity, affinity of thecopolymer to the surface of a suitable supporting substrate, such as acoating layer on the inner surface or exposed bare surface of acapillary tubing, and chirality. The preferred inherent properties ofthe secondary comonomers are hydrophilicity, hydrophobicity, viscosity,and self coating properties. The selection of the secondary comonomersand the ratio of secondary comonomers to primary comonomer are based onpredetermined desired properties of the targeted random comonomer.

At least one secondary comonomer may be copolymerized with the primarycomonomer to form a random copolymer, wherein each comonomer unit isdistributed along the copolymer chain in no specific order, and theratio of the reactivity of the primary comonomer to the secondarycomonomers is between about 0.3 to about 2. The reactivity is theprobability that a given monomer is added to a growing copolymer chainin the presence of other types of monomers. Formation of the randomcopolymers is not limited to the copolymerization of one secondarycomonomer with the primary comonomer. More than one secondary comonomermay be present in the formation of the random copolymers.

In another embodiment, the secondary comonomer or comonomers are vinylmonomers, monomers of acrylamide derivatives, monomers of acryloylderivatives, monomers of acrylic acid derivatives and mixtures thereof.Preferably, the secondary comonomers are vinyl monomers, monomers ofacrylamide derivatives, monomers of acryloyl derivatives, monomers ofacrylic acid derivatives, monomers of polyoxides, monomers ofpolysilanes, monomers of polyethers, monomers of derivatizedpolyethylene glycols, monomers of cellulose compounds, and mixturesthereof, each having between 2-24 carbon atoms, is saturated orunsaturated, and is substituted or unsubstituted.

More preferably, the secondary comonomer includes at least one ofmethacrylamide, N-acryloylmorpholine, N-allylacrylamide,N-allylmethacrylamide, N, benzylacrylamide, N-benzylmethacrylamide,N-(iso-butoxymethyl)acrylamide, N-(iso-butoxymethyl) methacrylamide,N-(tert-butyl)acrylamide, N-tert-butyl)methacrylamide,N-cyclohexylacrylamide, N-cyclohexylmethacrylamide,N,N-diethylacrylamide, N,N-diethylmethacrylamide,N-[2-(N,N-dimethylamino-)ethyl]acrylamide,N-[2-(N,N-dimethylamino)ethyl]methacrylamide,N-[3-(N,N-dimethylamino)propyl]acrylamide,N-[3-(N,N-dimethylamino)propyl-]methacrylamide, N,N-dimethylacrylamide,N-methylmethacrylamide, N-methylacrylamide, N-ethylacrylamide,N-ethylmethyacrylamide, N-phenylacrylamide, N-phenylmethacrylamide,N,N-diphenylacrylamide, N,N-diphenylmethacrylamide,N,N-dodecamethylenebisacrylamide, N-dodecylacrylamide,N-dodecylmethacrylamide, N-(2-hydroxypropyl)acrylamide,N-(2-hydroxypropyl)methacrylamide, N,N-methylenebismethacrylamide,N-methylolacrylamide, N-methylolmethacrylamide, N-propylacrylamide,N-propylmethacrylamide, N-isopropylacrylamide,N-isopropylmethacrylamide, N-butylacrylamide, N-butylmethacrylamide,N-isobutylacrylamide, N-isobutylmethacrylamide, vinyl acetate,vinylacetic acid, vinylbenzyl alcohol, vinylcyclohexane, N-vinylformamide, 1-vinyl-2-pyrrolidinone, vinyl acetonitrile, vinyl acrylate,vinyl 4-tert-butylbenzoate, N-vinylcaprolactam, vinyl crotonate,vinylcyclopentane, vinyl decanoate, vinyl carbonate, vinyl2-ethylhexanoate, 1-vinylimidazole, vinyl methacrylate,2-vinyinaphthalene, 2-vinylpyridine, 4-vinylpyridine, vinyl sulfone,ethylene glycol vinyl ether, 1,6-hexanediol vinyl ether,N-vinylphthalimide, vinyl pivalate, 1-vinyl-2-pyrrolidinone, vinyltrifluoroacetate, 4,4′-vinylidenebis(N,N-dimethylaniline), or mixturesthereof. In another embodiment, the secondary comonomer can also includeacrylamide alone or in combination with any of the above comonomers.

The random copolymers are synthesized by copolymerization of comonomersusing methodology well known to those of ordinary skill in the art. Thepreferred method of copolymer synthesis is free-radical solutionpolymerization. Any free radical initiator well known to those ofordinary skill in the art may be used, including, but not limited to,peroxy compounds, azoalkanes, photochemical homolysis, biradicals, tinhydrides, alkyl amines, and heat. Preferably, the free radical initiatoris a peroxy compound, an azoalkane, or alkylamine.

Typical polymerization initiators known to those of ordinary skill inthe art can be used in the present invention. For instance, theseinitiators may be capable of generating free radicals. Suitablepolymerization initiators include both thermal and photoinitiators.Suitable thermal initiators include, but are not limited to, ammoniumpersulfate/tetramethylethylene diamine, 2,2′-azobis-(2-amidino propane)hydrochloride, potassium persulfate/dimethylaminopropionitrile,2,2′-azobis(isobutyronitrile), 4,4′-azobis-(4-cyanovaleric acid), andbenzoyl-peroxide. Preferred thermal initiators are ammoniumpersulfate/tetramethyethylenediamine and 2,2′-azobisisobutyronitrile(“AIBN”). Suitable photoinitiators include, but are not limited to,isopropylthioxantone, 2-(2′-hydroxy-5′-methylphenyl)benzotriazole,2,2′-dihydroxy-4-methoxybenzophenone, and riboflavin. When using thecombination of persulfate and tertiary amine, the persulfate ispreferably added prior to the addition of the non-aqueous medium, sincepersulfate is much more soluble in water than in non-aqueous dispersingmedia. More preferably, the free radical initiator isN,N,N′,N′-tetramethylethylene-diamine (“TEMED”), or AIBN.

The matrix of the present invention has a higher long-term storagechemical stability because the matrix is at least essentially free ofdenaturing agents and detrimental degradation products therefrom. Thematrix of the present invention also has a higher thermal stability andcan be operated at higher temperatures to improve denaturing efficiencythan if denaturants were present in the matrix. Denaturing agents can bethermally unstable and produce degradation products detrimental to theperformance of the separation support and matrix.

A matrix of the present invention, which is essentially free ofdenaturing agents preferably has a lower viscosity than a matrixutilizing chemical denaturation, such as formamide, to achieve the samelevel of denaturation. This advantage is due at least in part to thehigher level of heating that can be obtained with the present matrices.The lower viscosity allows faster separation speeds than if denaturantswere used.

Advantages provided by running the essentially denaturant free matrix ofthe present invention also include, for example, an increased capillarylifetime, a longer sequencing read length, and a higher confidence levelof a called base, as measured by a phred score, which is describedbelow. For example, when a 5% 1:1 copolymer matrix containing 7M urea isused for room temperature electrophoresis, a capillary array lasts onlyup to 14 runs. A capillary comprising the essentially denaturant freematrix of the present invention lasts for more than about 25 runs. Thus,for example, at least about 25 samples can be run in sequence withoutproviding a new matrix within the separation support. The actual numberof runs that can be obtained with a single capillary before providing anew matrix depends, for example, upon the type of coating and theoperating temperature.

The 5% 1:1 copolymer essentially denaturant free matrix running at 80°C. has a longer sequencing read length than the same matrix with urearunning at about 20° C. This is an indication of the efficientdenaturing power achieved by running the matrix at 80° C. This isfurther validated by sequencing PGEM/U using universal M13 reverseprimer, which has been widely used as a control sample in commercial DNAsequencers. The PGEM/U sample running at 80° C. using the non-denaturantmatrix shows no sign of compression, whereas the same sample run in adenaturing matrix does show signs of compression.

Without urea or formamide present in the matrix, the matrix is morestable at elevated temperature. Preferably the matrix can be heated to atemperature sufficiently greater than the reannealing temperature of theDNA to disrupt the secondary structure of the DNA, which improves readlength Because separation is faster at higher temperatures, one canlower the running voltage to further extend the read length. The matrixof the present invention provides a read length greater than about 600base pairs and more preferably greater than about 650 base pairs.

A Phred score is a standard used widely in the sequencing community tomeasure the quality or confidence level of a called base. It is thenegative logarithm of the error probability of a called base. Forexample, a Phred score of 20 for a base means the probability of errorfor calling this base is 1/100 or 1%. A Phred score of 20 is a standardcut-off threshold used in popular sequencing facilities. Only thosebases with a Phred score greater than or equal to 20 are consideredreliable and can be accepted into downstream in a sequence assemblingprocess. As shown below, the present invention provides a higher phredscore than achieved by using a sieving matrix comprising a denaturantsuch as urea.

Device for Separations Utilizing Thermal Denaturation

FIG. 1 a shows a preferred arrangement of an embodiment of the presentthermal sequencing device 1. A sample capillary 3 is provided toelectrophoretically separate unknown sample compounds. As used herein,the term “capillary” collectively refers to any support or structureconfigured and arranged to separate a sample using electrophoresis.Thus, as used herein, the term “capillary” refers not only to what arecommonly called capillaries but to microfabricated channels, and planarstructures, such as used in slab gel electrophoresis. Capillary 3 ispreferably arranged to be in fluid contact with a sample reservoir 5,which is configured to contain a volume of sample sufficient to performan analysis. Examples of suitable sample reservoirs include the wells ofa microtitre plate, a structure configured to perform PCR amplificationon a volume of sample, a reservoir of a microfabricated electrophoresisdevice, and the like. Alternatively, where planar structures are used,an aliquot of sample can be added, such as by pipette, to the matrix.

Device 1 is provided with a power supply (not shown) suitable forproviding a sufficient voltage and current for electrophoreticseparation of a sample. The power supply is preferably configured toallow at least one of the current or resistance of the capillary to bemonitored during a separation. Preferably, the current or resistancedata is received by the computing device 17 to allow the electricpotential to be varied to maintain a constant current or resistance.

A temperature controlled portion 7 of sample capillary 3 is arranged tobe in thermal contact with a heat source such as a hot plate 9, or thelike. Optionally, or in addition, the external heat source may comprisea wire, filament, or other heating element arranged external to thecapillary. The capillary is preferably surrounded by a thermallyconductive medium 13, to enhance thermal contact between the heatingsource and the capillary.

During electrophoresis, the external heat source, rather than ohmicheating of the separation medium itself, is preferably the dominantsource of thermal energy to the separation medium within the capillary.The heat source is configured to heat the separation matrix to suitableoperational temperatures of the matrix, as discussed above. Thetemperature is preferably sufficient to substantially denature DNA inthe matrix without boiling a fluid in the matrix which is eitheressentially, or completely, free of denaturing agents. For example, theheat source is configured to heat the matrix to a temperature of atleast about 75° C. and preferably between 80° C.-99° C. More preferably,the temperature is between 80° C.-95° C. and most preferably is between80° C.-90° C.

The temperature of the capillary is monitored by a temperature sensingdevice in thermal contact with the separation support, such as athermocouple 15, which preferably sends data to a computing device 17.The temperature measured by the temperature sensing device is consideredto be the temperature of the separation matrix and the sample beingelectrophoresed. Hot plate 9 is preferably automatically controlled bycomputing device 17 in response to temperature data received fromsensing device 15. Thus, device 1 preferably includes computer meanscomprising at least one of software or a memory configured to operatethe heat source to heat the capillary to a temperature suitable forseparation of thermally denatured nucleic acids, as described above.

Device 1 also includes a light source 23, such as a laser emitting lighthaving a wavelength suitable to generate fluorescence from a fluorescentdye. A detector 25 is arranged to obtain fluorescence intensity data,such as a time-intensity electropherogram including peaks indicative ofthe presence of nucleotides, and send the detected fluorescenceintensities to computing device 17. A detection system such as thatdisclosed in U.S. Pat. No. 6,118,127, can be used for this purpose.

In any embodiment of the present invention, the fluorescence intensitydata of the unknown sample can be obtained simultaneously with thefluorescence intensity data of a second sample. By simultaneously, it ismeant that the unknown and second samples are elecrophoresed in a totaltime at least about 25% less, preferably about 50% less, than twice thetime required to sequentially electrophorese the samples. Preferably,the unknown sample is subjected to capillary electrophoresis in thesample capillary 3 and the second sample is simultaneously subjected tocapillary electrophoresis in a second, different capillary 19.

FIG. 1 b shows another embodiment of a thermal denaturation device 40 inwhich a temperature control zone 50 of the sample capillary 3 andoptional reference capillary 19 are placed in thermal contact with agas, such as air or nitrogen. Device 40 is provided with a power supply75, having the same features as the power supply of device 1 discussedabove. Temperature control zone 50 preferably extends for a heatedlength 64 of the capillaries. At least one inlet port 52 is provided tointroduce the heated gas to a region 54 between the capillaries and athermal jacket 56 and at least one outlet 58 is provided to allow thegas to exit. Thermal jacket 56 insulates temperature control zone 50 toreduce heat loss from the temperature control zone 50.

The gas can be heated using, for example, a resistively heated filament60 or a heat exchanger.

Preferably, a fan 62 or other device to force the gas into the inlet andout of the exit is provided. Use of a gas, which has a lower viscositythan other fluids such as liquids, allows the temperature of thecapillary to be changed much more rapidly because the temperature of thegas can be changed using, for example, a heated filament much morerapidly than that of a more viscous liquid. It should be understood,however, that a liquid may be used to thermostat the temperature of thetemperature control zone.

A cooled zone 80 having a second, cooled length 66 of capillaries 3 and19 can be provided to deliberately reduce the temperature of the samplesbeing separated after the samples have passed through the temperaturecontrol zone 50. In the context, ‘deliberate cooling’ means somethingother than simply allowing the matrix to cool by simply exposing thecapillary, microchip or slab to room temperature. The temperature incooled zone 80 can be controlled using chilled air or other fluid orliquid with an arrangement similar to that provided in the temperaturecontrol zone. Alternatively, a peltier cooler can be arranged in thermalcontact with this portion of the capillary, to reduce the temperature.The temperature and length 66 of cooled zone 80 are preferably lowenough and long enough, respectively, to allow a DNA fragment that wasthermally denatured within temperature control zone 50 to anneal priorto being detected at a reference detection zone 70 or a sample detectionzone 70′. Thus, the cooled zone is configured and disposed to receivecompounds that have migrated electrophoretically through heated zone 50of the capillary. The temperature is reduced to less than about 45° C.,more preferably to less than about 30° C., and most preferably to lessthan about 20° C.

Example

The invention is further illustrated through the following non-limitingexample.

FIGS. 2 a and 2 b show the result of sequencing a PGEM/U sample using a5% copolymer matrix in 1xTBE, 7M urea, at about 20° C. The copolymer waspolymerized using a 1:1 ratio of acrylamide and N,N-dimethylacrylamide(DMA) monomer.

As can be seen in FIG. 3, which shows the graph of Phred score versuscalled bases for FIGS. 2 a and 2 b, from base #25 to base #405, thecalled bases satisfy the criteria of Phred score 20. We define thesection of the base sequence with Phred score greater than or equal to20 as the trim length. For the example in FIG. 3, the trim length is405−25=380. The trim length is used as a measurement of the matrixperformance.

FIGS. 4 a and 4 b show the result of sequencing a PGEM/U sample using 5%copolymer gel in 1xTBSE, at 80° C. according to the present invention.No urea was used. The copolymer was polymerized from 1:1 acrylamide andDMA monomer. FIG. 5 shows the Phred score v. called bases. The trimlength for this example is 720−34−686, which is an improvement over 380.

The present invention also provides a higher separation speed thanelectrophoresis using a chemical denaturant. For the example in FIGS. 2a and 2 b using urea gel at room temperature, it takes 120 minutes for500 bases to pass the detector. FIGS. 4 a and 4 b, however, show thatusing non-urea gel at 80° C. according to the invention, it takes 68minutes for 500 bases to pass the detector, and 96 minutes for 800 basesto pass the detector. Overall, tremendous gain in separation speed andqualified sequencing length has been demonstrated by using the non-ureagel at 80° C.

While the above invention has been described with reference to certainpreferred embodiments, it should be kept in mind that the scope of thepresent invention is not limited to these. Thus, one skilled in the artmay find variations of these preferred embodiments which, nevertheless,fall within the spirit of the present invention, whose scope is definedby the claims set forth below.

1. A method of separating a first sample comprising nucleic acids, themethod comprising: providing a matrix that is essentially free ofdenaturing agents; raising a temperature of a first portion of thematrix to at least about 80° C.; subjecting the nucleic acids toelectrophoresis through at least the first portion of the matrix whilethe temperature of the first portion is at least about 80° C.; anddeliberately cooling a second portion of the matrix to less than about30° C., the nucleic acids migrating through the second portion afterthey have first migrated through the first portion.
 2. The method ofclaim 1, wherein the first portion of the matrix is raised to atemperature between 80° C.-90° C.
 3. The method of claim 1, wherein thematrix comprises at least one random, linear copolymer comprising afirst comonomer of acrylamide and at least one secondary comonomer. 4.The method of claim 1, wherein the second portion of the matrix iscooled to less than about 25° C.
 5. The method of claim 1, wherein thematrix is completely free of denaturing agents.
 6. The method of claim1, further comprising subjecting a second sample of nucleic acids toelectrophoresis within the same matrix, after the first sample has beenelectrophoresced.
 7. The method of claim 6, comprising subjecting atotal of at least 25 additional samples of nucleic acids, one at a time,without replacing the matrix.
 8. The method of claim 7, wherein thetemperature of at least a portion of the polymer matrix in which thesecond sample is electrophoresced is at least about 80° C.
 9. A methodof separating a first sample comprising nucleic acids, the methodcomprising: subjecting the nucleic acids to electrophoresis using amatrix that is essentially free of denaturants, the matrix having atleast one random, linear copolymer comprising a first comonomer ofacrylamide and at least one secondary comonomer, wherein a temperatureof at least a portion of the matrix is at least about 80° C.
 10. Themethod of claim 9, wherein the comonomers are randomly distributed alongthe copolymer, and wherein the at least one secondary comonomer isselected from the group consisting of vinyl monomers, monomers ofacrylamide derivatives, monomers of acryloyl derivatives, monomers ofacrylic acid derivatives, monomers of polyoxides, monomers ofpolysilanes, monomers of polyethers, monomers of derivatizedpolyethylene glycols, monomers of cellulose compounds, or mixturesthereof, each having between 2-24 carbon atoms.
 11. The method of claim9, wherein the at least one secondary comonomer isN,N-dimethylacrylamide monomer.
 12. The method of claim 11, wherein thepolymer is a copolymer polymerized using about a 1:1 ratio of acrylamideand N,N-dimethylacrylamide monomer.
 13. A method of sequencing a samplecomprising nucleic acids, comprising: providing a matrix that isessentially free of denaturing agents, the matrix having at least onerandom, linear copolymer comprising about a 1:1 ratio of acrylamide andN,N-dimethylacrylamide monomer, and a buffer having a pH of at leastabout 8, a temperature of at least a portion of the matrix being atleast about 80° C.; subjecting the nucleic acids to electrophoresisthrough said matrix; and prior to detecting the nucleic acids,deliberately cooling a second portion of the matrix to less than about25° C., the second portion of the matrix receiving nucleic acids fromthe heated portion of the matrix.
 14. A method of separating a pluralityof samples of biological compounds, comprising: providing a matrix thatis essentially free of denaturing agents; subjecting a first sample toelectrophoresis through said matrix, the first sample comprising nucleicacids, and wherein a temperature of a first portion of the matrix issufficient to substantially denature the nucleic acids; and subjecting asecond sample to electrophoresis in a separate step but through the samematrix, the second sample comprising a complex of at least twobiological compounds.
 15. The method of claim 14, wherein thetemperature is from about 80° C. to about 99° C.
 16. The method of claim15, wherein the temperature is from about 80° C. to about 90° C.
 17. Themethod of claim 15, further comprising deliberately cooling a secondportion of the matrix to less than about 30° C., the first and secondsamples migrating through the second portion after each has firstmigrated through the first portion.
 18. The method of claim 17, whereinthe second portion of the matrix is cooled to less than about 25° C. 19.The method of claim 15, wherein the complex comprises at least one of anucleic acid-protein complex and a protein-protein complex.